The invention relates generally to methods for generating modified nucleic acid molecules, and more particularly, to the use of polymerase chain reaction to generate gene knockouts and nucleic acid fusion molecules.
There are a variety of reasons which make the modification of nucleic acid sequences, particularly genes, desirable. The classical strategy for gene disruption requires the isolation of a gene and digestion with restriction enzymes [R. Rothstein, Methods Enzymol., 101:202-211 (1983)]. However, the use of restriction enzymes to digest the DNA fragments sometimes makes it difficult to construct appropriate fragments disrupted by a marker DNA. To overcome this problem, several methods utilizing polymerase chain reaction (PCR) for constructing such deletions have been developed. However, it is still necessary to isolate the DNA fragment of interest, or to use a variety of complex steps [D. C Amberg et al, Yeast, 11:1275-1280 (1995); A. Wach etal, Yeast, 10:1793-1808 (1994); A. Wach, Yeast, 12:259-265 (1996)].
One recently described method describes a strategy for constructing gene disruption cassettes by means of PCR and ligation. See, J. Nikawa and M. Kawabat, Nucleic Acids Res., 26(3):860-861 (1998). In a first step, two separate regions of a target gene are PCR amplified with primers specific for the target sequence and genornic DNA as a template. Secondly, the two PCR products are ligated with a DNA fragment of a marker gene through two separate reactions. The ligated fragments are then PCR amplified separately. Following amplification the PCR amplified fragments are mixed, denatured, annealed, and extended with DNA polymerase. Finally, the product is PCR amplified with the outermost primers.
Despite these recent advances, there remains a need for methods for modifying nucleic acid molecules which are more efficient, yet permit precise engineering at the target site.
The method of the invention provides a simple method for precisely generating a modified nucleic acid molecule to contain a deletion and/or an insertion. Advantageously, this method does not require ligation and is well suited for use in automated formats, including high throughput formats.
In one aspect the invention provides a three-stage method for inserting a cassette into a nucleic acid molecule to produce a modified nucleic acid molecule fusion without requiring ligation. In the first stage, the method involves amplifying two separate regions of a selected nucleic acid molecule and a cassette. The two regions of the nucleic acid molecule have nucleotide sequences flanking a site in the molecule targeted for disruption, whereby the amplification produces a first amplification product of nucleotide sequences upstream of the target site and a second amplification product of nucleotide sequences downstream of the target site. The cassette has sequences at its 5xe2x80x2 and 3xe2x80x2 ends which overlap with sequences of the two regions of the nucleic acid molecule. In the second stage, the amplified cassette product is mixed separately with the first or second amplification products resulting from amplification of the nucleic acid molecule. The cassette and first amplification product are amplified by PCR, thereby forming a first fusion product consisting of the first amplification product fused to the 5xe2x80x2 end of the first strand of the cassette. The cassette is also mixed with the second amplification product and this mixture is amplified to form a second fusion product consisting of the second amplification product fused to the 3xe2x80x2 end of the first strand of the cassette. In a third stage, the first and second fusion products are mixed and amplified by PCR, thereby producing a modified nucleic acid molecule comprising the cassette in the target site of the selected nucleic acid molecule. Desirably, the resulting modified nucleic acid molecule is amplified via polymerase chain reaction.
In another aspect, the invention provides a novel method for performing amplifying selected sequences by PCR, which is particularly well suited for use in the stage three of the method of the invention. In this method, a mixture containing the fusion products prepared according to stage 2 of the method of the invention is heated for about 5 minutes in the absence of polymerase or primers at about 94xc2x0 C., cooled to 50xc2x0 C. over about 30 minutes, at which temperature it is maintained for about 5 minutes or longer. A thermostable polymerase is then added to the mixture, which is heated to about 72xc2x0 C. for about 5 minutes, and mixed with a forward primer P1 for the first region and a reverse primer P4 for the second region. The resulting mixture is then amplified using PCR to produce a modified nucleic acid molecule comprising the first and second regions of the nucleic acid sequence flanking the cassette.
In a further aspect, the invention provides a two stage method of producing a modified nucleic acid moleucle without ligation. The method involves producing two separate regions of a nucleic acid molecule and a cassette as in stage I of the three stage method of the invention. Thereafter, the three products are mixed and subjected to amplification by PCR, as described in the aspect above. Thus, this embodiment of the invention permits elimination of stage 2 of the three-stage method.
In yet a further aspect, the invention provides modified nucleic acid sequences produced using the method of the invention.
In yet another aspect, the present invention provides a method of high throughput preparation of disrupted Streptococcus DNA sequences without ligation. This method involves mixing (a) a nucleic acid molecule comprising Streptococcus DNA sequences comprising a first region upstream of a site in the Streptococcus DNA targeted for disruption and a second region downstream of the target site, said first and second region each having a first and second end, (b) a cassette comprising at one end, nucleotide sequences which overlap with nucleotides at the second end of the first region, and at its other end, nucleotides which overlap with nucleotides of the first end of the second region, and (c) primers for the first and second regions in each of the wells of a plate containing a plurality of reaction wells. This mixture is then subjected to PCR, thereby amplifying the first and second regions of the selected Streptococcus DNA sequences. The cassette and the amplified first and second regions of the Streptococcus DNA sequences are then mixed and subjected to polymerase chain reaction to produce a nucleic acid fusion molecule comprising the first and second regions of the Streptococcus DNA sequence flanking the cassette.
Other aspects and advantages of the invention will be readily apparent from the detailed description of the invention.